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Common fragile sites (CFSs) are loci that are hypersensitive to replication stress and hotspots for chromosomal rearrangements in cancers. CFSs replicate late in S phase, are cell-type specific and nest in large genes. The relative impact of transcription–replication conflicts versus a low density in initiation events on fragility is currently debated. Here we addressed the relationships between transcription, replication, and instability by manipulating the transcription of endogenous large genes in chicken and human cells. We found that inducing low transcription with a weak promoter destabilized large genes, whereas stimulating their transcription with strong promoters alleviated instability. Notably, strong promoters triggered a switch to an earlier replication timing, supporting a model in which high transcription levels give cells more time to complete replication before mitosis. Transcription could therefore contribute to maintaining genome integrity, challenging the dominant view that it is exclusively a threat.

One of the most fascinating features of biological systems is the ability to sustain high accuracy of all major cellular processes despite the stochastic nature of underlying chemical processes. It is widely believed that such low error values are the result of the error-correcting mechanism known as kinetic proofreading. However, it is usually argued that enhancing the accuracy should result in slowing down the process, leading to the so-called speed–accuracy trade-off. We developed a discrete-state stochastic framework that allowed us to investigate the mechanisms of the proofreading using the method of first-passage processes. With this framework, we simultaneously analyzed the speed and accuracy of the two fundamental biological processes, DNA replication and tRNA selection during the translation. The results indicate that these systems tend to optimize speed rather than accuracy, as long as the error level is tolerable. Interestingly, for these processes, certain kinetic parameters lay in the suboptimal region where their perturbations can improve both speed and accuracy. Additional constraints due to the energetic cost of proofreading also play a role in the error correcting process. Our theoretical findings provide a microscopic picture of how complex biological processes are able to function so fast with high accuracy.

Replication timing (RT) is a cellular program to coordinate initiation of DNA replication in all origins within the genome. RIF1 (replication timing regulatory factor 1) is a master regulator of RT in human cells. This role of RIF1 is associated with binding G4-quadruplexes and changes in 3D chromatin that may suppress origin activation over a long distance. Many effects of RIF1 in fork reactivation and DNA double-strand (DSB) repair (DSBR) are underlined by its interaction with TP53BP1 (tumor protein p53 binding protein). In G1, RIF1 acts antagonistically to BRCA1 (BRCA1 DNA repair associated), suppressing end resection and homologous recombination repair (HRR) and promoting non-homologous end joining (NHEJ), contributing to DSBR pathway choice. RIF1 is an important element of intra-S-checkpoints to recover damaged replication fork with the involvement of HRR. High-resolution microscopic studies show that RIF1 cooperates with TP53BP1 to preserve 3D structure and epigenetic markers of genomic loci disrupted by DSBs. Apart from TP53BP1, RIF1 interact with many other proteins, including proteins involved in DNA damage response, cell cycle regulation, and chromatin remodeling. As impaired RT, DSBR and fork reactivation are associated with genomic instability, a hallmark of malignant transformation, RIF1 has a diagnostic, prognostic, and therapeutic potential in cancer. Further studies may reveal other aspects of common regulation of RT, DSBR, and fork reactivation by RIF1.

Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe . The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.

R-loops are three-stranded structures that harbour an RNA–DNA hybrid and frequently form during transcription. R-loop misregulation is associated with DNA damage, transcription elongation defects, hyper-recombination and genome instability. In contrast to such ‘unscheduled’ R-loops, evidence is mounting that cells harness the presence of RNA–DNA hybrids in scheduled, ‘regulatory’ R-loops to promote DNA transactions, including transcription termination and other steps of gene regulation, telomere stability and DNA repair. R-loops formed by cellular RNAs can regulate histone post-translational modification and may be recognized by dedicated reader proteins. The two-faced nature of R-loops implies that their formation, location and timely removal must be tightly regulated. In this Perspective, we discuss the cellular processes that regulatory R-loops modulate, the regulation of R-loops and the potential differences that may exist between regulatory R-loops and unscheduled R-loops.R-loops (three-stranded RNA–DNA structures) are often associated with transcription defects, DNA damage and genome instability, but ‘regulatory’ R-loops can promote gene regulation, telomere stability and DNA repair. This dual functionality of R-loops requires tight control of their formation, location and timely removal.

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors. PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.

Accurate replication of DNA requires stringent regulation to ensure genome integrity. In human cells, thousands of origins of replication are coordinately activated during S phase, and the velocity of replication forks is adjusted to fully replicate DNA in pace with the cell cycle 1 . Replication stress induces fork stalling and fuels genome instability 2 . The mechanistic basis of replication stress remains poorly understood despite its emerging role in promoting cancer 2 . Here we show that inhibition of poly(ADP-ribose) polymerase (PARP) increases the speed of fork elongation and does not cause fork stalling, which is in contrast to the accepted model in which inhibitors of PARP induce fork stalling and collapse 3 . Aberrant acceleration of fork progression by 40% above the normal velocity leads to DNA damage. Depletion of the treslin or MTBP proteins, which are involved in origin firing, also increases fork speed above the tolerated threshold, and induces the DNA damage response pathway. Mechanistically, we show that poly(ADP-ribosyl)ation (PARylation) and the PCNA interactor p21 Cip1 (p21) are crucial modulators of fork progression. PARylation and p21 act as suppressors of fork speed in a coordinated regulatory network that is orchestrated by the PARP1 and p53 proteins. Moreover, at the fork level, PARylation acts as a sensor of replication stress. During PARP inhibition, DNA lesions that induce fork arrest and are normally resolved or repaired remain unrecognized by the replication machinery. Conceptually, our results show that accelerated replication fork progression represents a general mechanism that triggers replication stress and the DNA damage response. Our findings contribute to a better understanding of the mechanism of fork speed control, with implications for genomic (in)stability and rational cancer treatment. Inhibition of PARP is shown to accelerate the speed of replication fork elongation, which prevents fork stalling and induces DNA damage, with implications for genomic instability and cancer treatment.

DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and recombination of the viral genome, and viral gene transcription. HSV-1 encodes its own DNA replication machinery, including an origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), and a helicase/primase complex (UL5/UL8/UL52). In addition, HSV-1 utilizes a combination of accessory viral and cellular factors to coordinate viral DNA replication with other viral and cellular processes. The purpose of this review is to outline the roles of viral and cellular proteins in HSV-1 DNA replication and replication-coupled processes, and to highlight how HSV-1 may modify and adapt cellular proteins to facilitate productive infection.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2 -deficient cells from DNA damage and rescues the lethality of Brca2 -deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2 -deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance. Protection of nascent DNA from degradation provides a mechanism that can promote synthetic viability and drug resistance in Brca -deficient cells without restoring homologous recombination at double-strand breaks. Chemoresistance in BRCA cancers The breast cancer susceptibility genes BRCA1 and BRCA2 function to protect the genome from DNA damage. For this reason, DNA-damaging agents are used clinically to treat BRCA -deficient cancers. However, these treatments may have a short window of effectiveness; many cancers develop resistance. André Nussenzweig and colleagues show that cells become drug resistant due to loss of the PTIP protein. In its absence, forks that stall during DNA replication are protected from degradation, and this allows the cells to survive. This work highlights a previously unknown mechanism by which resistance to cancer therapy can arise.

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5′ ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3′ ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.